This project describes the development of photoswitchable enediynes, which are stable in the dark but convert into very reactive cyclic enediyne compounds upon irradiation with light. The latter then undergoes facile cycloaromatization to produce a p-benzyne diradical. The photo-induced formation of highly strained enediynes opens the opportunity for the spatial and temporal control of the generation of the cytotoxic p-benzyne and direct spectroscopic observation of its reactions. The photoswitchable enediynes will be used to study the reversibility of the Bergman cyclization, as well as the feasibility of the enediyne reactivity control by shifting the position of keto-enol equilibrium. The photo-precursor of the azo-analog of the activated neocarzinostatin chromophore will be prepared to explore the azo-Myers cycloaromatization. The photoswitchable enediynes suitable for the two-photon activation will be developed as well. The non-resonant two-photon excitation (2PE) allows for the use of light in a so-called "phototherapeutic window", a region of relative tissue transparency between 650 and 950 nm, as well as conduct irradiation through a UV-Vis opaque media. In addition, the 2PE provides an opportunity for three-dimensional control of enediyne reactivity. To achieve these goals, photoprecursors of highly strained nine-membered ring enediynes, as well as cyclic enediynes incorporating additional endocyclic double bonds, will be synthesized. The photoswitchable enediynes containing extended chains of conjugation with D-A-D and D-pi-A-pi-D structural motifs will be used for the 2PE experiments. The in-situ generation of reactive enediynes is based on two strategies: 1) the photochemical ring contraction of eleven-membered ring enediynes and 2) the in situ photochemical generation of pi-components of the enediyne conjugated system. The successful completion of this project will supply new tools for exploring the mode of action of natural antitumor antibiotics. Photoactivatable enediynes can serve as a basis for the development of new photoactivatable enediyne antibiotics and 3-D selective photonucleases.